The differentiation and the functional development of male germ cells remain pooriy understood at a molecular level, with many of the crucial regulators of these critical processes yet to be identified. While the general structure of the "9+2" axoneme critical to sperm flagellar function has been established by investigation in organisms such as Chlamydomonas reinhardtii, the mammalian orthologues of known critical genes are only now being uncovered. This research draws on the existent knowledge of "9+2" cilia to pinpoint both conserved and proprietary structures and mechanisms in the mammalian system, with the aim of improving understanding of male fertility at the critical level of sperm motility and of furthering discernment of the "9+2" axoneme structure in general. The overarching hypothesis of the proposed research is that the murine orthologue ofthe central axoneme component PF20, Spag16 plays a bi-functional role in the development of motile sperm via production of two distinct proteins, with SPAG16L serving as an essential structural element and SPAG16S as a modulator of gene expression within the spermiogenic network. As the SPAG16S transcript is believed to be testis specific, it is hypothesized that the protein's role in gene expression reflects the unique requirements of male germ cell development, and that elucidation of SPAG16S targets and interactors may lead to the discovery of other critical players in determining male fertility. The hypotheses to be tested in the proposed research include: 1) that deletion of Spag16S results in male non-transmission ofthe affected allele;2) that deletion of Spag16S produces defects in spermatogenesis such that transgenic males will exhibit impaired sperm motility, aberrant sperm development and function;3) that the role of Spag16S is testis-specific, and that its deletion will be inconsequential in females and in non-testis male tissue;4) that Spag16S exerts its influence by modulation of gene expression, and that Spag16S deletion will result in measurable changes in levels of both transcripts and proteins;5) that Spag16S acts in a specific manner vi targeted interaction with effector proteins. Public Health Relevance: Human reproduction and its regulation are critical societal and public health concerns, which encompass the treatment of infertility, 40% of which is caused by a male factor, and contraception. The proposed research explores fundamental aspects ofthe process of spermatogenesis with the goal of elucidating novel regulatory mechanisms that will inform the understanding of male infertility and potentially disclose new male germ cell-specific targets for contraception.